Horseradish peroxidase labeled streptavidin biotin method

AApoAII and AA fibrils were also detected by immunohistochemistry (IHC) with specific rabbit antiserum against mouse ApoA-II or mouse AA, which were produced against guanidine hydrochloride-denatured AApoAII or AA fibrils in our laboratory [20 (link), 21 (link), 33 (link), 38 (link)]. Four-micron (4 μm) thick sections of fixed organs were treated with 3% H₂O₂ in methanol for 30 minutes (min) to inactivate endogenous peroxidase and were blocked with 5% bovine serum albumin in PBS. The sections were incubated overnight at 4°C with rabbit antisera against mouse ApoA-II (1 : 3000) and AA (1 : 3000) prepared in our laboratory [39 (link)] or 4-hydroxynonenal (4-HNE) (1 : 300, Abcam plc, Cambridge, UK), followed by incubation with the biotinylated secondary antibody (Abcam plc). Target proteins were identified by the horseradish peroxidase-labeled streptavidin-biotin method (DAKO, Glostrup, Denmark). In a negative control section, the first antibody was omitted to confirm the specificity of staining. To analyze the positive area in each organ quantitatively, the ratios of the positively stained area to a whole organ in randomly captured 5 areas under ×200 or ×400 magnification were measured using image processing program (NIH ImageJ software, version 1.61) [33 (link)].

We detected AApoAII deposition and catalase by immunohistochemistry (IHC) following a previously described method (Li et al., 2017 (link)). Antiserum against mouse ApoA-II was produced against guanidine hydrochloride-denatured AApoAII in our laboratory (Higuchi et al., 1983 (link)) and applied at a dilution ratio of 1:3000. Catalase antibody was applied (1:500, GTX110704, GeneTex Inc, CA, USA) to reveal the degree of peroxisome change in the liver. After incubation overnight at 4°C with the primary antibody, the sections were incubated with the biotinylated secondary antibody (1:300, DAKO, Glostrup, Denmark) for 1 hr at room temperature. Target proteins were identified by the horseradish peroxidase-labeled streptavidin-biotin method (1:300, DAKO). In the immunofluorescence experiments, the sections were incubated with the PPARα antibody (1:500, GTX101098, GeneTex Inc, CA, USA) overnight and incubated with Alexa Fluor 488 goat anti-rabbit antibody (1:500, Thermo Fisher Scientific, Japan) for 1 hr at room temperature and incubated with DAPI for 10 min. Images were captured immediately using a confocal laser fluorescence microscope (LSM 880 with Airyscan, Carl Zeiss, Germany). In a negative control section, the primary antibody was omitted to confirm the specificity of staining.